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8. Restriction modification.ppt

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MSU & Skol. Tech Restriction/modification MSU & Skol. Tech Restriction/modification

Restriction/modification Restriction endonucleases cleave DNA at specific sites Restriction/modification Restriction endonucleases cleave DNA at specific sites

Restriction/modification Methyltransferases methylate specific DNA sites thus preventing DNA cleavage by restriction endonucleases Restriction/modification Methyltransferases methylate specific DNA sites thus preventing DNA cleavage by restriction endonucleases

Restriction/modification protects bacteria from phage attacks Restriction/modification protects bacteria from phage attacks

Restriction/modification Methylation is maintained through cell divisions, permanently protecting host DNA from cleavage Restriction/modification Methylation is maintained through cell divisions, permanently protecting host DNA from cleavage

Restriction/modification R/E systems could also be considered as selfish elements, since they kills bacteria, Restriction/modification R/E systems could also be considered as selfish elements, since they kills bacteria, which lost the corresponding genes

Restriction/modification Restriction endonuclease types Restriction/modification Restriction endonuclease types

Restriction/modification Restriction endonuclease types Restriction/modification Restriction endonuclease types

Restriction/modification Methyltransferases utilize SAM as a methyl group donor Restriction/modification Methyltransferases utilize SAM as a methyl group donor

Restriction/modification Methyltransferases usually contains conserved catalytic domain and variable recognition domain Restriction/modification Methyltransferases usually contains conserved catalytic domain and variable recognition domain

Restriction/modification C-methyltransferases use a stepwise mechanism via covalent intermediate Restriction/modification C-methyltransferases use a stepwise mechanism via covalent intermediate

Restriction/modification Regulation of R/E systems Aims of regulation 1. Methyltransferase should appear first to Restriction/modification Regulation of R/E systems Aims of regulation 1. Methyltransferase should appear first to protect host DNA 2. Restriction nuclease expression should follow that of methyltransferase 3. In a steady state methyltransferase shouldn’t be in a too much excess (not to methylate phage DNA)

Restriction/modification Regulation of Cfr. BI Before DNA methylation After DNA methylation Restriction/modification Regulation of Cfr. BI Before DNA methylation After DNA methylation

Restriction/modification Regulation of Cfr. BI Before DNA methylation After DNA methylation Restriction/modification Regulation of Cfr. BI Before DNA methylation After DNA methylation

Restriction/modification Regulation of Sso. II Before C protein Medium level of C protein Excess Restriction/modification Regulation of Sso. II Before C protein Medium level of C protein Excess of C protein

Restriction/modification Homing endonucleases Restriction/modification Homing endonucleases

Restriction/modification Meganucleases Restriction/modification Meganucleases

Restriction/modification Restriction endonuclease genes as mobile elements? Restriction/modification Restriction endonuclease genes as mobile elements?

Restriction/modification Phage defense against restriction Restriction/modification Phage defense against restriction

CRISPR (clustered regularly interspaced short palindromic repeats)-associated system (CRISPR-Cas) Spacers contains sequences from phage CRISPR (clustered regularly interspaced short palindromic repeats)-associated system (CRISPR-Cas) Spacers contains sequences from phage and plasmids Acquisition of a spacer makes cell resistant to the phage containing the corresponding DNA

CRISPR Expression of CRISPR/Cas locus lead to the formation of defence complex Cascade CRISPR Expression of CRISPR/Cas locus lead to the formation of defence complex Cascade

CRISPR Cascade recognize and cleaves invader DNA that match cr. RNA CRISPR Cascade recognize and cleaves invader DNA that match cr. RNA

CRISPR Cascade recognizes and cleaves target DNA CRISPR Cascade recognizes and cleaves target DNA